The present invention, in some embodiments thereof, relates to chemical conjugates and to uses thereof and, more particularly, but not exclusively, to polymeric conjugates having attached thereto a therapeutically active agent and an additional therapeutically active agent and/or a targeting moiety, to processes of preparing such conjugates and to uses thereof.
A polymeric drug delivery system can be designed for passive or active targeting of tumors. Passive targeting refers to the exploitation of the natural (passive) distribution pattern of a drug-carrier in vivo. The latter is based upon the phenomenon named the “enhanced permeability and retention (EPR) effect”, and attributed to two factors: (I) the disorganized pathology of angiogenic tumor vasculature with its discontinuous endothelium, leading to hyperpermeability to circulating macromolecules, and (II) the lack of effective tumor lymphatic drainage, which leads to subsequent macromolecular accumulation. The active approach relies upon the selective localization of a ligand at a cell-specific receptor.
A well-designed polymeric drug delivery system, whether it is targeting the tumor site passively or actively, improves the therapeutic index of anti-angiogenic and chemotherapeutic agents by increasing the half-life of low molecular weight drugs, their selective tumor accumulation, their water-solubility and their time of exposure to the tumor vasculature (i.e. to the tumor endothelial cells), while reducing their toxicity.
Angiogenesis is a biological process that involves the sprouting of new blood vessels from pre-existing ones and plays a crucial role in disease development and progression. Pathological angiogenesis has been demonstrated in several diseases, including atherosclerosis, cancer, hypertension, rheumatoid arthritis, diabetes and diabetes related complications such as diabetic retinopathy.
Since tumor growth and metastasis are particularly dependent on the degree of angiogenesis, many drugs have been developed, which target different steps in the multi-step tumor angiogenesis process. However, most of these drugs were shown to be cytostatic rather than cytotoxic and thus do not cause a substantial reduction of tumor volume during the first stage of treatment. Currently approved anti-cancer therapies with recognized anti-angiogenic properties mainly include monoclonal antibodies directed against specific pro-angiogenic factors and/or their receptors (e.g., Avastin, Erbitux, Vectibix, Herceptin); small molecule tyrosine kinase inhibitors (TKIs) of multiple pro-angiogenic growth factor receptors (e.g., Tarceva, Nexavar, Sutent, Iressa); and inhibitors of mTOR (mammalian target of rapamycin) (e.g., Torisel).
Neural cell adhesion molecule (NCAM, CD56) is a cell adhesion molecule structurally belonging to the immunoglobulin superfamily. It is expressed on most brain tumors. In several other tumor types, NCAM expression was shown to be associated with more aggressive biological behavior, increased metastatic capacity and expression of stem-cell markers. NCAM was found to be expressed on tumor endothelial cells, but not on normal endothelial cells (Bussolati et al., Exp Cell Res, 2006). Recently, it was proven that in Wilms' tumor, a common pediatric solid malignancy of the kidney, the cancer stem cells (CSC) population is uniquely characterized by the expression of NCAM (Pode-Shakked et al., J Cell Mol Med, 2008). CSC have been characterized by the expression of NCAM in other cancers as well, including hepatocellular carcinoma, hepatoblastoma and lung carcinoma (Fiegel et al., J Histochem Cytochem, 2004, Xu et al., Carcinogenesis, 2009). Therefore, NCAM provides a specific biomarker than can be exploited to target the cancer stem cells and tumor endothelial cells.
The microtubule-interfering agent Paclitaxel (PTX) is a clinically well-established and highly-effective anti-neoplastic drug used as a monotherapy and in combination therapy mainly for the treatment of prostate, breast, ovarian, and non-small cell lung cancers and it is the drug of choice for the treatment of metastatic breast cancer. It has also shown anti-angiogenic and pro-apoptotic properties [Oldham et al. 2000 Int. J Oncol. 16:125-132]. However, due to the hydrophobic nature of the drug, solubilizing agents such as Cremophor EL or ethanol are required for its administration. PTX causes severe adverse side effects such as neutropenia, neuropathies, and when solubilized in Cremophor EL causes hypersensitivity reactions. In addition, only a small amount of the drug localizes in the tumor and the drug is substrate to efflux pumps in particular p-glycoprotein, resulting in multiple drug resistance.
Doxorubicin (DOX) is one of the most potent antineoplastic drugs prescribed alone or in combination with other agents, remaining the compound of its class that has the widest spectrum of activity. Doxorubicin is commonly used in the treatment of Wilms' tumor, as well as various solid tumors and hematological malignancies. Although DOX is recognized as a potent antineoplastic agent, its cardiotoxic effects are the main reason for the dose limited administration. Other common side effects associated with its use are acute nausea and vomiting, stomatitis, gastrointestinal disturbances, alopecia, baldness, neurologic disturbances and bone marrow aplasia.
Conjugation of anti-cancer drugs to copolymers, such as HPMA copolymer or PGA, has been suggested so as to restrict the passage through the blood brain barrier and to prolong the circulating half-life of the drugs, hence inhibiting the growth of tumor endothelial and epithelial cells by exposing the cells to the conjugated drugs in the circulation for a longer time compared to the free drugs.
U.S. Pat. No. 6,884,817 teaches compositions comprising a chemotherapeutic and/or anti-angiogenic drug, conjugated to a water-soluble polyamino acid or soluble metal chelator.
The conjugate paclitaxel-polyglutamate OPAXIO™ (paclitaxel poliglumex, CT-2103) (Formerly known as XYOTAX™) showed promising results in phase III trials and is currently being evaluated for marketing approval.
U.S. patent application Ser. No. 12/117,678 having Publication No. 2008/0279778 also teaches polyglutamate polymers conjugated to a plurality of drugs for use in drug targeting, stabilizing and imaging applications. A HPMA copolymer conjugate of paclitaxel has also been described by Meerum Terwogt et al. [Anticancer drugs 2001; 12:315-323].
WO 03/086382 teaches conjugates of water-soluble polymers and the anti-angiogenic agent TNP-470, and their use as anti-tumor agents, in particular their use as carriers of TNP-470 into tumor vessels, and their effect on the neurotoxicity of TNP-470. WO 2006/084054 teaches that an HPMA copolymer-TNP-470 conjugate (caplostatin) can be used in combination with an anti-EGF monoclonal antibody for treating an angiogenic disease.
WO 03/086178 teaches a method for decreasing or inhibiting disorders associated with vascular hyperpermeability by the administration of an effective amount of an anti-angiogenic compound or a compound capable of increasing cell-cell contacts by stabilizing tight junction's complexes and increasing contact with the basement membrane. According to the teachings of WO 03/086178, HPMA copolymer-TNP-470 inhibited vascular endothelial growth factor (VEGF)-induced vessel hyperpermeability and inhibited angiogenesis both in vitro and in vivo.
Integrins are a class of receptors involved in the mechanism of cell adhesion. Since the 1980s it is well recognized that integrins play a key role in cell matrix interactions and hence in angiogenesis.
The integrins are heterodimeric transmembrane glycoproteins that compose a diverse family of 19 α and eight β subunits. An integrin with a well-characterized involvement in angiogenesis and tumor invasiveness is αvβ3. αvβ3 integrins are known to bind the RGD sequence (Arg-Gly-Asp), which constitutes the recognition domain of different proteins, such as laminin, fibronectin and vitronectin. The RGD sequence represents the minimal amino acid domain, in several extracellular matrix proteins, which has been demonstrated to be the binding site of the transmembrane integrins proteins family [Bazzoni et al. 1999, Current Opinion in Cell Biology; (11) pp. 573-581].
It has been demonstrated that RGD-containing peptides, either isolated from phage peptides library or biochemically synthesized, were able to compete with extracellular matrix proteins on binding to integrins [Haubner et al. 1997, Angew. Chem. Int. Ed. Engl.; (36) pp. 1374-1389]. Tumor-induced angiogenesis can be targeted in vivo by antagonizing the αvβ3 integrin with small peptides containing the RGD amino acid sequence.
It has been further found that the substrate specificity of RGD-containing peptides results from the different conformations of the RGD sequence in different matrix proteins. For example, the bis-cyclic peptide E-[c(RGDfK)2] is a ligand-based vascular-targeting agent that binds to integrin αvβ3 (Eldar-Boock et al, Biomaterials 32(15):3862-3874, 2011).
Chen et al. reported [J. Med. Chem. 2005; 48:1098-1106] the synthesis and antitumor activity of paclitaxel (PTX) conjugated with a bis-cyclic RGD (E[RGDyK]2) in a metastatic breast cancer cell line.
Mitra et al. report [Journal of Controlled Release 2006; 28: 175-183] the biodistribution and tumor targeting properties of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer based conjugates of mono-(RGDfK) and doubly cyclized (RGD4C) αvβ3 binding peptides.
WO 2006/012355 teaches an anti-angiogenic polymer conjugate for treatment of solid tumors comprising a chemical moiety targeting cell-surface proteins of endothelial cells at an angiogenic site. The chemical moiety taught in the application may be a ligand such as RGD4C or RGDfK for a cell-surface receptor, such as, for example, an integrin. The polymer conjugate taught by WO 2006/012355 may further comprise at least one side chain comprising a chelator capable of chelating a pharmaceutically acceptable radioactive label. Wan et al. [2003 Proc. Int'l Symp. Control. Rel. Bioact. Mater. Vol 30: 491-492] teach targeting endothelial cells using HPMA copolymer-doxorubicin-RGD conjugates.
Bisphosphonates, such as alendronate, are molecules used to treat osteoporosis, bone metastases and to prevent bone fractures. These compounds exhibit an exceptionally high affinity to the bone mineral hydroxyapatite, and therefore are known to be used also as a targeting moiety (Uludag, H. Curr Pharm Des 2002; 8: 1929-1944).
Alendronate is considered potent for the treatment of bone related diseases and cancer-associated hypercalcemia. It was shown to have antitumor effect in several in vivo cancer models through several different mechanisms [Tuomela et al. 2008, BMC Cancer 8:81; Molinuevo et al. 2007, Eur J Pharmacol 562:28-33; Hashimoto et al. 2005, Cancer Res 65: 540-545]. In addition, alendronate was found to have anti-angiogenic activity through (i) suppression of VEGF-induced Rho activation in an ovarian cancer model [Hashimoto et al. 2007, Biochem Biphys Res Commun 354: 478-484], (ii) inhibition of farnesyl pyrophosphate synthase, in the mevalonate pathway [Russell RG 2007, Pediatrics 119 Suppl 2: S150-162]; and (iii) regulation of cellular level of MMP-2 expression in osteosarcoma cell lines [Cheng et al. 2004, Pediatr Blood Cancer 42; 410-415].
WO 2004/062588 teaches water soluble polymeric conjugate for bone targeted drug delivery with improved pharmacokinetics parameters and better water solubility of the loaded drugs. The polymeric drug delivery systems taught by this application are based on hydroxypropyl methacrylate (HPMA) copolymer conjugates of bone-targeting drugs such as alendronate and D-Asp8 together with a bone-related therapeutic agent (e.g., tetracycline).
PK2 (FCE28069) is a HPMA copolymer-doxorubicin-galactosamine conjugate, which was designed as a treatment for hepatocellular carcinoma or secondary liver disease [Seymour et al. Journal of Clinical Oncology 2002; 20: 1668-1676]. Galactosamine binds to the hepatic asialoglycoprotein receptor (ASGPR) thus serving as a specific hepatic targeting moiety. These components are linked to the HPMA copolymer via an enzymatically-biodegradable linker which permits the release of free doxorubicin within the liver, thus increasing the drug concentration in its site of action. The enzymatic degradable linker is a tetrapeptide spacer (Gly-Phe-Leu-Gly; GFLG), designed for cleavage by lysosomal cathepsins.
O'hare et al. [Journal of Drug Targeting 1993; 1:217-229] have synthesized HPMA copolymers containing doxorubicin and melanocyte stimulating hormone (MSH) as a melanoma specific targeting moiety. Both the doxorubicin and the melanocyte stimulating hormone were linked to the HPMA polymer via an enzymatically biodegradable linker.
Hruby et al. [Journal of Applied Polymer Science 2006; 101:3192-3201] have synthesized novel polymeric drug-delivery systems designed for bone targeting of anti-neoplastics based on biocompatible HPMA copolymers containing hydroxybisphosphonate targeting moieties and the model drugs radiotherapeutics 125I, imaging agent 111In, or the anticancer drug Doxorubicin.
WO 2009/141823 discloses conjugates of polymers or copolymers (e.g., HPMA) having attached thereto an anti-angiogenic agent (e.g., PTX) and a bisphosphonate bone targeting agent such as alendronate, and uses thereof.
WO 2009/141827 discloses conjugates of hydroxypropyl methacrylamide (HPMA) copolymer-derived copolymers having attached thereto TNP-470 and a high load (e.g., higher than 3 mol %) of alendronate (ALN), prepared by RAFT polymerization.
WO 2009/141826 discloses conjugates of a polymer (e.g., PGA) having attached thereto an angiogenesis targeting moiety (e.g., RGD-containing peptide) and an anti-cancer agent or anti-angiogenic agent (e.g., PTX), and uses thereof.
The teachings of WO 2009/141823, WO 2009/141826 and WO 2009/141827 are incorporated by reference as if fully set forth herein.
The “reversible addition-fragmentation chain transfer” (RAFT) polymerization technique typically involves the use of thiocarbonylthio compounds, such as dithioesters, dithiocarbamates, trithiocarbonates, and xanthates in order to mediate the polymerization via a reversible chain-transfer process. This allows access to polymers with low polydispersity and high functionality.
Additional background art includes Satchi-Fainaro et al., 2002, Bioorganic & Medicinal Chemistry, 10 (9), 3023-3029; Marsili et al., 2008, Peptides 29, 2232-2242; Segal et al. PLoS One 2009, 4(4):e5233; Eldar-Boock et al. Biomaterials 2011, 32(15):3862-3874; Pan et al., Biomacromolecules. 2011; 12(1):247-52.